Integration of meningococcal conjugate vaccination

ABSTRACT

Conjugated meningococcal capsular saccharides will be introduced into immunization schedules in the near future, but the phenomenon of “carrier suppression” must first be addressed, particularly where multiple conjugates are to be used. It has been found that diphtheria toxoid and its derivatives (such as CRM197) can safely be used as the carrier protein, even where multiple meningococcal conjugates are administered at the same time and where a patient has previously been exposed to the carrier protein, either in the form of a previous immunogen (e.g. in a DTP vaccine) or as a previous carrier protein (e.g. in a Hib or pneumococcal conjugate vaccine). The invention provides a method for immunizing a patient, comprising administering multiple conjugates of meningococcal capsular saccharides, wherein each conjugate comprises a diphtheria toxoid (or derivative thereof) carrier protein, and the capsular saccharide, and wherein the patient has been pre-immunized with a diphtheria toxoid (or derivative thereof).

All documents cited herein are incorporated by reference in theirentirety.g

RELATED APPLICATIONS

This application is the U.S. National Phase of International ApplicationNo. PCT/GB2005/001653, filed Apr. 29, 2005 and published in English,which claims priority to Great Britain Application No. 0409750.7, filedApr. 30, 2004 and Great Britain Application No. 0500787.7, filed Jan.14, 2005. The teachings of the above applications are incorporatedherein in their entirety by reference.

TECHNICAL FIELD

This invention concerns vaccines against Neisseria meiningitidis. Inparticular, it concerns vaccines based on conjugated capsularsaccharides from multiple meningococcal serogroups.

BACKGROUND ART

Based on the organism's capsular polysaccharide, twelve serogroups of N.meningitidis have been identified (A, B, C, H, I, K, L, 29E, W135, X, Yand Z). Group A is the pathogen most often implicated in epidemicdisease in sub-Saharan Africa. Serogroups B and C are responsible forthe vast majority of cases in USA and in most developed countries.Serogroups W135 and Y are responsible for the remaining cases in USA anddeveloped countries.

A tetravalent vaccine of capsular polysaccharides from serogroups A, C,Y and W135 has been known for many years [1,2]. Although effective inadolescents and adults, it induces a poor immune response and shortduration of protection and cannot be used in infants [e.g. ref. 3]because polysaccharides are T cell-independent antigens that induce aweak immune response which cannot be boosted. The polysaccharides inthis vaccine are not conjugated [4].

Conjugate vaccines against serogroup C have been approved for human use,and include Menjugate™ [5], Meningitec™ and NeisVac-C™. Mixtures ofconjugates from serogroups A+C are known [6-8] and mixtures ofconjugates from serogroups A+C+W135+Y have been reported [9-13].

While meningococcal conjugates are well known, they have not yet beenfitted into existing pediatric immunisation schedules, which fordeveloped countries typically involve: hepatitis B vaccine at birth;and, starting at 2 months, all of diphtheria/tetanus/pertussis (D-T-P),H. influenzae type, b (Hib) conjugate, inactivated poliovirus andpneumococcus conjugates at 2 months.

When adding conjugated vaccines to existing immunisation schedules,however, the issue of carrier-induced epitopic suppression (or “carriersuppression”, as it is generally known) must be addressed, particularlysuppression arising from carrier priming. “Carrier suppression” is thephenomenon whereby pre-immunisation of an animal with a carrier proteinprevents it from later eliciting an immune response against a newantigenic epitope that is presented on that carrier [14].

As reported in reference 15, where several vaccine antigens contain thesame protein component (being used as an immunogen and/or as a carrierprotein in a conjugate) then there is the potential for interferencebetween those antigens. In reference 15, the immune response against anantigen that was conjugated to a tetanus toxoid (Tt) carrier wassuppressed by pre-existing immunity against Tt.

Reference 16 reports how a combination of D-T-P vaccines with a Hibconjugate vaccine was adversely affected where the carrier for the Hibconjugate was the same as the tetanus antigen from the D-T-P vaccine.The authors concludes that this “carrier suppression” phenomenon,arising from interference by a common protein carrier, should be takeninto account when introducing vaccines that include multiple conjugates.

In contrast to references 15 and 16, reference 17 reported that primingwith tetanus toxoid had no negative impact on the immune responseagainst a subsequently-administered Hib-Tt conjugate, but suppressionwas seen in patients with maternally acquired anti-Tt antibodies. Inreference. 18, however, an “epitopic suppression” effect was reportedfor a Tt-based peptide conjugate in patients having existing anti-Ttantibodies resulting from tetanus vaccination.

In reference 19, it was suggested that a conjugate having CRM197 (adetoxified mutant of diphtheria toxin) as the carrier may be ineffectivein children that had not previously received diphtheria toxin as part ofa vaccine (e.g. as part of a D-T-P or D-T vaccine). This work wasfurther developed in reference 20, where a carrier priming effect by D-Timmunisation was seen to persist for subsequent immunisation with Hibconjugates.

In reference 21, the authors found that pre-immunisation with adiphtheria or tetanus toxoid carrier protein reduced the increase inanti-Hib antibody levels after a subsequent immunisation with the Hibcapsular saccharide conjugated to those carriers, with IgG1 and IgG2being equally affected. Responses to the carrier portions of theconjugates were also suppressed. Furthermore, a more generalnon-epitope-specific suppression was seen, as pre-immunisation with oneconjugate was seen to affect immune responses against both the carrierand saccharide portions of a second conjugate that was administered fourweeks later.

The use of different carrier proteins in a single multivalentpneumococcal conjugate vaccine is reported in reference 22, withmultiple carriers being used in order to avoid carrier suppression. Theauthors predict that there is a maximum load of a carrier protein thatcan be tolerated in a multivalent conjugate vaccine without giving riseto negative interference. In reference 23 it was reported thatpneumococcal conjugate vaccines including mixed carrier proteinselicited, in parallel to the anti-pneumococcus response, unintentionalbooster responses to the carriers.

In reference 24, an investigation of whether diphtheria and tetanusboosters could be administered with monovalent meningococcal serogroup Cconjugates, it was found that titres against the meningococcal conjugatewere reduced where the carrier was tetanus toxoid carrier and thepatient had received prior immunisation with a tetanus-containingvaccine.

Finally, reference 25 reports that “prior exposure to the carrierprotein can either enhance or suppress antibody response topolysaccharides administered in saccharide-protein conjugates”. Theconjugates used in reference 25 used tetanus toxoid or the CRM197 mutantas carrier protein.

The situation concerning carrier priming and/or suppression is thusconfused, and it remains unclear whether any particular conjugate willsuffer from carrier suppression or will benefit from a carrier primingenhancement. Meningococcal conjugate vaccines will not be in a positionto be integrated into or added to existing pediatric immunisationschedules until this issue is addressed. Furthermore, as meningococcalconjugates are to be administered as tetravalent mixtures (i.e. fourdifferent conjugates) then the potential for carrier suppression becomeseven more of a risk.

In addition to the problem of priming with a carrier having a negativeimpact on immune responses against saccharide conjugates, the reversecan also occur i.e. immunisation with a conjugate can have a negativeimpact on immune responses against the carrier [26].

DISCLOSURE OF THE INVENTION

Reference 27 suggests that carrier suppression in meningococcalconjugate vaccines should be dealt with by using more than one type ofcarrier protein. In particular, reference 27 suggests that H. influenzaeprotein D should be used as the carrier protein for meningococcalconjugates, with tetanus toxoid (Tt) also being a possibility. To avoidepitope suppression, protein D is also the carrier of choice inreference 28. Similarly, reference 29 suggests that Bordetella pertussisfimbriae should be used as the carrier in order to avoid carriersuppression in multivalent conjugate vaccines. In contrast, it has beenfound that diphtheria toxoid (Dt) and its derivatives can safely be usedas the carrier for meningococcal saccharide conjugates, even wheremultiple meningococcal conjugates are administered at the same time.None of the monovalent meningococcal serogroup C conjugates studied inreference 24 used a Dt carrier.

Moreover, reference 27 also suggests that meningococcal conjugatevaccines should be administered at the same time as D-T-P-Hib vaccines(e.g. see example 3), such that there is no previous exposure to thecarrier protein from the meningococcal conjugates. In contrast, it hasnow been found that meningococcal conjugates can be administered topatients even where they have already received the carrier protein,either in the form of a previous immunogen (e.g. in a D-T-P or a D-Timmunisation) or as a previous carrier protein (e.g. in a Hib conjugateor pneumococcal conjugate vaccine): The previous study ofcarrier-induced epitopic suppression in monovalent serogroup C conjugatevaccines [24] did not look at the effect of any prior administration ofconjugates.

As well as contrasting with reference 27, the ability of a patient toraise an immune response against a meningococcal conjugate, even wherethey have already received a different conjugate, contrasts withreference 21.

Thus the invention provides a method for immunising a patient against adisease caused by Neisseria meningitidis, comprising the step ofadministering to the patient a composition that comprises at least twoof: (a) a conjugate of (i) the capsular saccharide of serogroup A N.meningitidis and (ii) a diphtheria toxoid or derivative thereof; (b) aconjugate of (i) the capsular saccharide of serogroup C N. meningitidisand (ii) a diphtheria toxoid or derivative thereof; (c) a conjugate of(i) the capsular saccharide of serogroup W135 N. meningitidis and (ii) adiphtheria toxoid or derivative thereof; and (d) a conjugate of (i) thecapsular saccharide of serogroup Y N. meningitidis and (ii) a diphtheriatoxoid or derivative thereof, wherein the patient has been pre-immunisedwith (a) a diphtheria toxoid or derivative thereof and/or (b) aconjugate of (i) a capsular saccharide of an organism other than N.meningitidis and (ii) a diphtheria toxoid or derivative thereof.

The invention also provides the use of at least two of: (a) a conjugateof (i) the capsular saccharide of serogroup A N. meningitidis and (ii) adiphtheria toxoid or derivative thereof; (b) a conjugate of (i) thecapsular saccharide of serogroup C N. meningitidis and (ii) a diphtheriatoxoid or derivative thereof; (c) a conjugate of (i) the capsularsaccharide of serogroup W135 N. meningitidis and (ii) a diphtheriatoxoid or derivative thereof; and (d) a conjugate of (i) the capsularsaccharide of serogroup Y N. meningitidis and (ii) a diphtheria toxoidor derivative thereof, in the manufacture of a medicament for immunisinga patient against a disease caused by Neisseria meningitidis, whereinthe patient has been pre-immunised with (a) a diphtheria toxoid orderivative thereof and/or (b) a conjugate of (i) a capsular saccharideof an organism other than N. meningitidis and (ii) a diphtheria toxoidor derivative thereof.

The meningococcal disease is preferably meningitis, more preferablybacterial meningitis, and most preferably meningococcal meningitis. Thusthe invention can be used to protect against meningococcal infectionsthat cause meningitis.

Where the pre-immunisation antigen is a derivative of a diphtheriatoxoid then that derivative preferably remains immunologicallycross-reactive with Dt, and is preferably CRM197.

The Pre-Immunised Patient

The patient to be immunised has been pre-immunised with: (a) adiphtheria toxoid or derivative thereof; and/or (b) a conjugate of (i) acapsular saccharide of an organism other than Neisseria meningitidis and(ii) a diphtheria toxoid or derivative thereof. Typical pre-immunisationwill have included: a diphtheria toxoid antigen; a Hib capsularsaccharide conjugate using a diphtheria toxoid or CRM197 carrier; and/ora pneumococcal capsular saccharide conjugate using a diphtheria toxoidor CRM197 carrier.

The patient will have received at least one (e.g. 1, 2, 3 or more) doseof the pre-immunisation antigen(s), and that dose (or the earliest ofmultiple doses) will have been administered to the patient at least six(e.g. 6, 9, 12, 15, 18, 21, 24, 36, 48, 60, 120, 180, 240, 300 or more)months before the immunisation with the meningococcal conjugatesaccording to the invention. In a preferred group of patients, thepre-immunisation took place within 3 years of birth e.g. within 2 yearsof birth, within 1 year of birth, within 6 months of birth, or evenwithin 3 months, 2 months or 1 month of birth.

The patient to be immunised according to the invention will typically bea human. The human will generally be at least 1 month old e.g. at least2 months old, at least 3 months old, at least 4 months old, at least 6months old, at least 2 years old, at least 5 years old, at least 11years old, at least 17 years old, at least 40 years old, at least 55years old, etc. A preferred set of patients is at least 6 months old.Another preferred set of patients is in the age group 2-55 years old,and another preferred set of patients is in the age group 11-55 yearsold. A further preferred set of patients is less than 11 years old e.g.2-11 years old. In all cases, however, regardless of age, the patientwill have been pre-immunised as defined herein.

Where the pre-immunisation antigen is a diphtheria toxoid then thepatient will typically have received the toxoid as the ‘D’ antigen in aD-T-P or a D-T pre-immunisation. Such immunisations are typically givento newborn children at ages 2, 3, and 4 months. Where the immunisationincludes a pertussis vaccine, that vaccine may be a whole cell orcellular pertussis vaccine (‘Pw’), but is preferably an acellularpertussis vaccine (‘Pa’). Pre-immunisation Pa vaccines will generallyinclude one, two or three of the following well-known andwell-characterised B. pertussis antigens: (1) pertussis toxoid (‘PT’),detoxified either by chemical means or by site-directed mutagenesis e.g.the 9K/129G′ mutant [30]; (2) filamentous haemagglutinin (‘FHA’); (3)pertactin (also known as ‘69 kiloDalton outer membrane protein’).Acellular pertussis vaccines may also include agglutinogen 2 and/oragglutinogen 3. The ‘T’ antigen in a D-T-P pre-immunisation is typicallya tetanus toxoid.

Where the pre-immunisation antigen is a diphtheria toxoid then thepatient may also or alternatively have received the toxoid as thecarrier protein of a protein-saccharide conjugate. Such conjugatesinclude the ‘PRP-D’ Hib conjugate [see Table 14-7 of ref. 32] e.g. theProHIBIT™ product.

Where the pre-immunisation antigen is CRM197 then the patient willtypically have been pre-immunised with a Hib conjugate and/or amultivalent pneumococcal conjugate. Such immunisations are typicallygiven to newborn children at ages 2, 3, and 4 months. Hib conjugatesthat use a CRM197 carrier include the ‘HbOC’ conjugates [Table 14-7 ofref. 32] e.g. the HibTITER™ product. Pneumococcal conjugates that use aCRM197 carrier include the 7-valent PCV7 mixtures e.g. the PrevNar™vaccine [31]. The patient may also have been pre-immunised with aserogroup C meningococcal (‘MenC’) conjugate. MenC conjugates that useCRM197 carrier include Meninvact™/Menjugate™ [5] and Meningitec™.Preferably, however, the patient has been pre-immunised with Hib and/orpneumococcal conjugate, but not with a MenC conjugate. If the patienthas been pre-immunised with a MenC conjugate then the vaccineadministered according to the invention may or may not include aserogroup C conjugate.

Where pre-immunisation was with a conjugated antigen then the patientwill almost inevitably have also received a small amount of freediphtheria toxoid (or derivative) as a result of low-level contaminationof the conjugate (e.g. caused by hydrolysis of the conjugate duringstorage), but this small amount will not typically have been adequate toprovide a significant immune response.

Diphtheria toxoid is a well known and well characterised protein [e.g.see chapter 13 of ref. 32] that can be obtained by treating theADP-ribosylating exotoxin of Corynebacterium diphtheriae with aninactivating chemical, such as formalin or formaldehyde. CRM197 is alsowell known and well characterised [33-36], and has been widely used as acarrier in conjugated saccharide vaccines. CRM197 and Dt share manycarrier epitopes.

The result of the pre-immunisation is that the patient's immune systemhas been exposed to the pre-immunisation antigens. For pre-immunisationwith diphtheria toxoid (Dt), this generally means that the patient willhave raised an anti-Dt antibody response (typically to give an anti-Dttiter >0.01 IU/ml) and will possess memory B and/or T lymphocytesspecific for Dt i.e. pre-immunisation with Dt is typically adequate toelicit an anamnestic anti-Dt immune response in the patient. Forpre-immunisation where Dt (or derivative) is a carrier for a saccharidewithin a conjugate then the pre-immunisation will have raised ananti-saccharide response and the patient will possess memory B and/or Tlymphocytes specific for the saccharide i.e. the pre-immunisation istypically adequate to elicit an anamnestic anti-saccharide immuneresponse in the patient. The pre-immunisation was preferably adequate toelicit protective immunity in the patient e.g. against diphtheriadisease.

Thus the patients to be immunised according to the invention aredistinct from patients in general, as they are members of a subset ofthe general population whose immune systems have already mounted animmune response to the pre-immunisation antigens, such that immunisationaccording to the invention with a meningococcal conjugate that includesa diphtheria toxoid (or derivative thereof) carrier elicits a differentimmune response in the subset than in patients who have not previouslymounted an immune response to the pre-immunisation antigens. Patientswho have been pre-immunised with Dt (or derivative) as the carrier of aconjugate (particularly of a Hib conjugate) are preferred. Particularlypreferred patients have been pre-immunised with Dt (or derivative) asthe carrier of a conjugate and also with Dt as an unconjugatedimmunogen.

As well as having been pre-immunised with a diphtheria toxoid (orderivative), in conjugated or non-conjugated form, the patient may havebeen pre-immunised with other antigens. Such antigens include, but arenot limited to: pertussis antigen(s)—see above; tetanus toxoid—seeabove; Haemophilus influenzae type B—see above; hepatitis B surfaceantigen (HBsAg); poliovirus, such as an inactivated poliovirus vaccine(IPV); Streptococcus pneumoniae—see above; influenza virus; BCG;hepatitis A virus antigens; measles virus; mumps virus; rubella virus;varicella virus; etc.

The patient may or may not have been pre-immunised with one or moremeningococcal conjugate(s). In some preferred embodiments, at the timewhen a patient first receives a meningococcal conjugate, they havealready been pre-immunised with Dt (or derivative). In otherembodiments, a meningococcal conjugate is administered to a patient whohas already been pre-immunised with both (i) Dt or a derivative and (ii)a meningococcal conjugate.

The Conjugates

The invention immunises patients with conjugated saccharides.Conjugation is used to enhance the immunogenicity of saccharides, as itconverts them from T-independent antigens to T-dependent antigens, thusallowing priming for immunological memory. Conjugation is particularlyuseful for pediatric vaccines [e.g. ref. 37] and is a well knowntechnique [e.g. reviewed in refs. 38 to 46].

The composition used according to the invention comprises at least twomeningococcal conjugates, wherein each conjugate comprises a diphtheriatoxoid (or derivative thereof) carrier protein, and the capsularsaccharide. The capsular saccharides are chosen from meningococcalserogroups A, C, W135 and Y, such that the compositions includesaccharides from 2, 3, or all 4 of these four serogroups. Specificcompositions comprise saccharides from: serogroups A & C; serogroups A &W135; serogroups A & Y; serogroups C & W135; serogroups C & Y;serogroups W135 & Y; serogroups A & C & W135; serogroups A & C & Y;serogroups A & W135 & Y; serogroups C & W135 & Y; serogroups A & C &W135 & Y. Compositions including saccharides from all four serogroupsare most preferred.

The capsular saccharides of each of these four serogroups are wellcharacterised. The capsular saccharide of serogroup A meningococcus is ahomopolymer of (α1→6)-linked N-acetyl-D-mannosamine-1-phosphate, withpartial O-acetylation in the C3 and C4 positions. The acetyl groups canbe replaced with blocking groups to prevent hydrolysis [47], and suchmodified saccharides are still serogroup A saccharides within themeaning of the present invention. The serogroup C capsular saccharide isa homopolymer of (α2→9)-linked sialic acid (N-acetyl neuraminic acid, or‘NeuNAc’). Most serogroup C strains have O-acetyl groups at C-7 and/orC-8 of the sialic acid residues, but about 15% of clinical isolates lackthese O-acetyl groups [48,49]. The saccharide structure is written as→9)-Neu p NAc 7/8OAc-(α2→. The serogroup W135 saccharide is a polymer ofsialic acid-galactose disaccharide units. Like the serogroup Csaccharide, it has variable O-acetylation, but at sialic acid 7 and 9positions [50]. The structure is written as:→4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Gal-α-(1→. The serogroup Y saccharide issimilar to the serogroup W135 saccharide, except that the disacchariderepeating unit includes glucose instead of galactose. Like serogroupW135, it has variable O-acetylation at sialic acid 7 and 9 positions[50]. The serogroup Y-structure is written as:→4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Glc-α-(1→.

The saccharides used according to the invention may be O-acetylated asdescribed above (e.g. with the same O-acetylation pattern as seen innative capsular saccharides), or they may be partially or totallyde-O-acetylated at one or more positions of the saccharide rings, orthey may be hyper-O-acetylated relative to the native capsularsaccharides.

The saccharides used according to the invention are preferably shorterthan the native capsular saccharides seen in bacteria. Thus thesaccharides are preferably depolymerised, with depolymerisationoccurring after purification but before conjugation. Depolymerisationreduces the chain length of the saccharides. A preferreddepolymerisation method involves the use of hydrogen peroxide [9].Hydrogen peroxide is added to a saccharide (e.g. to give a final H₂O₂concentration of 1%), and the mixture is then incubated (e.g. at about55° C.) until a desired chain length reduction has been achieved.Another depolymerisation method involves acid hydrolysis [10]. Otherdepolymerisation methods are known to the skilled person. Thesaccharides used to prepare conjugates for use according to theinvention may be obtainable by any of these depolymerisation methods.Depolymerisation can be used in order to provide an optimum chain lengthfor immunogenicity and/or to reduce chain length for physicalmanageability of the saccharides.

Typical carrier proteins for use in conjugates are bacterial toxins ortoxoids, such as diphtheria toxin (or its CRM₁₉₇ mutant) and tetanustoxin. Other known carrier proteins include the N. meningitidis outermembrane protein, synthetic peptides, heat shock proteins, pertussisproteins, cytokines, lymphokines, hormones, growth factors, artificialproteins comprising multiple human CD4⁺ T cell epitopes from variouspathogen-derived antigens, protein D from H. influenzae, pneumococcalsurface protein PspA, iron-uptake proteins, toxin A or B from C.difficile, etc. According to the invention, however, the meningococcalconjugates include a diphtheria toxoid (or derivative thereof, such asCRM197) carrier protein. Covalent conjugation is preferred.

It is possible to use more than one carrier protein in the compositions.Thus different carrier proteins can be used for different serogroupse.g. serogroup A saccharides might be conjugated to CRM197 whileserogroup C saccharides might be conjugated to diphtheria toxoid. It isalso possible to use more than one carrier protein for a particularsaccharide antigen e.g. serogroup A saccharides might be in two groups,with some conjugated to CRM197 and others conjugated to diphtheriatoxoid. In general, however, it is preferred to use the same carrierprotein for all meningococcal saccharides in the composition, and morepreferably for all saccharides (i.e. including any non-meningococcalconjugates that may be present). It is preferred that compositions ofthe invention do not include any tetanus toxoid carrier protein. Wherethe composition includes a diphtheria toxoid carrier protein then it ispreferred that it does not include any CRM197 carrier protein.

A single carrier protein might carry more than one saccharide antigen[51]. For example, a single carrier protein might have conjugated to itsaccharides from serogroups A and C. To achieve this goal, saccharidescan be mixed prior to the conjugation reaction. In general, however, itis preferred to have separate conjugates for each serogroup. Conjugatesare preferably mixed to give substantially a 1:1:1:1 ratio (measured asmass of saccharide) e.g. the mass of each serogroup's saccharide iswithin ±10% of each other. A typical quantity of meningococcal antigenper serogroup in a composition is between 1 μg and 20 μg e.g. between 2and 10 μg per serogroup, or about 4 μg. As an alternative to a 1:1:1:1ratio, a double serogroup A dose may be used (2:1:1:1).

Conjugates with a saccharide:protein ratio (w/w) of between 1:15 (i.e.excess protein) and 15:1 (i.e. excess saccharide), preferably between1:10 and 10:1, more preferably between 1:5 and 5:1, are preferred.Excess carrier protein is preferred. Conjugates with saccharide:proteinratio of about 1:12 or about 1:6 or about 1:3 are preferred,particularly where the carrier is Dt. A 1:3 ratio is most preferred.

Conjugates may be used in conjunction with free carrier protein [52].When a given carrier protein is present in both free and conjugated formin a composition of the invention, however, the unconjugated form ispreferably no more than 5% of the total amount of the carrier protein inthe composition as a whole, and more preferably present at less than 2%by weight. Similarly, unconjugated saccharide is preferably no more than15% by weight of the total amount of saccharide.

Any suitable conjugation reaction can be used, with any suitable linkerwhere necessary.

The saccharide will typically be activated or functionalised prior toconjugation. Activation may involve, for example, cyanylating reagentssuch as CDAP (e.g. 1-cyano-4-dimethylamino pyridinium tetrafluoroborate[53, 54, etc.]). Other suitable techniques use carbodiimides,hydrazides, active esters, norborane, p-nitrobenzoic acid,N-hydroxysuccinimide, S-NHS, EDC, TSTU; see also the introduction toreference 44).

Linkages via a linker group may be made using any known procedure, forexample, the procedures described in references 55 and 56. One type oflinkage involves reductive amination of the polysaccharide, coupling theresulting amino group with one end of an adipic acid linker group, andthen coupling a protein to the other end of the adipic acid linker group[42, 57, 58]. Other linkers include B-propionamido [59],nitrophenyl-ethylamine [60], haloacyl halides [61], glycosidic linkages[62], 6-aminocaproic acid [63], ADH [64], C₄ to C₁₂ moieties [65] etc.As an alternative to using a linker, direct linkage can be used. Directlinkages to the protein may comprise oxidation of the polysaccharidefollowed by reductive amination with the protein, as described in, forexample, references 66 and 67.

A process involving the introduction of amino groups into the saccharide(e.g. by replacing terminal ═O groups with —NH₂) followed byderivatisation with an adipic diester (e.g. adipic acidN-hydroxysuccinimido diester) and reaction with carrier protein ispreferred.

In one preferred conjugation method, a saccharide is reacted with adipicacid dihydrazide. For serogroup A, carbodiimide may also be added atthis stage. After a reaction period, sodium cyanoborohydride is added.Derivatised saccharide can then be prepared e.g. by ultrafiltration. Thederivatized saccharide is then mixed with carrier protein (e.g. with adiphtheria toxoid), and carbodiimide is added. After a reaction period,the conjugate can be recovered. Further details of this conjugationmethod can be found in reference 10. Conjugates obtainable by thismethod are preferred conjugates for use according to the invention e.g.conjugates comprising a diphtheria toxoid carrier and an adipic acidlinker.

Conjugates are preferably prepared separately and then mixed. Aftermixing, the concentration of the mixed conjugates can be adjusted e.g.with sterile pyrogen-free, phosphate-buffered saline. Each conjugate,before mixing, preferably contains no more than 15 μg of carrier.

The result of administering meningococcal conjugates according to theinvention is preferably that, for each administered serogroup, thepatient raises a serum bactericidal antibody (SBA) response, with theincrease in SBA titre (compared to the pre-immunised patient beforereceiving the mixed meningococcal conjugates) being at least 4-fold, andpreferably at least 8-fold. The SBA test is a standard correlate formeningococcal protection. Further details of serologic correlates formeningococcal vaccines are given in reference 68.

Further Antigenic Components of Compositions Used According to theInvention

In addition to meningococcal conjugates, compositions used according tothe invention may optionally include 1, 2 or 3 of the following furtherantigens:

-   1. A conjugated capsular saccharide from S. pneumoniae [e.g. chapter    23 of ref. 32; refs. 69-71].    -   It is preferred to include saccharides from more than one        serotype of S. pneumoniae. For example, mixtures of        polysaccharides from 23 different serotype are widely used, as        are conjugate vaccines with polysaccharides from between 5 and        11 different serotypes [72]. For example, PrevNar™ [31] contains        antigens from seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F)        with each saccharide individually conjugated to CRM197 by        reductive amination, with 2 μg of each saccharide per 0.5 ml        dose (4 μg of serotype 6B), and with conjugates adsorbed on an        aluminium phosphate adjuvant. Where pneumococcal conjugates are        included in a compositions for use with the invention, the        composition preferably includes at least serotypes 6B, 14, 19F        and 23F.-   2. A conjugated capsular saccharide from H. influenzae B [e.g.    chapter 14 of ref. 32].    -   The carrier protein for the conjugate may be CRM197, Dt, a        tetanus toxoid or an outer membrane complex of N. meningitidis.        The saccharide moiety of the conjugate may be a polysaccharide        (e.g. full-length polyribosylribitol phosphate (PRP)), but it is        preferred to depolymerise the capsular polysaccharides to form        oligosaccharides (e.g. MW from ˜1 to ˜5 kDa). A preferred Hib        conjugate comprises an oligosaccharide covalently linked to        CRM197 via an adipic acid linker [73,74]. Administration of the        Hib antigen preferably results in an anti-PRP antibody        concentration of >0.15 μg/ml, and more preferably >1 μg/ml.        Where a composition includes a Hib saccharide antigen, it        preferably does not also include an aluminium hydroxide        adjuvant. If the composition includes an aluminium phosphate        adjuvant then the Hib antigen may be adsorbed to the adjuvant        [75] or it may be non-adsorbed [27]. Prevention of adsorption        can be achieved by selecting the correct pH during        antigen/adjuvant mixing, an adjuvant with an appropriate point        of zero charge, and an appropriate order of mixing for the        various different antigens in a composition [76].-   3. A Protein Antigen from Neisseria meningitidis Serogroup B [e.g.    Ref. 77].

The composition may comprise one or more of these further antigens.

Such antigens may or may not be adsorbed to an aluminium salt.

If meningococcal conjugates are being administered in a series of dosesthen none, some or all of the doses may include these extra antigens.

Compositions containing the meningococcal conjugates preferably do notinclude tetanus toxoid. They preferably do not include pertussisantigens. They preferably do not include hepatitis B virus surfaceantigen. They preferably do not include poliovirus. A compositionpreferably contains no more than 50 μg of diphtheria toxoid permeningococcal conjugate, and more preferably no more than 50 μg ofdiphtheria toxoid for all meningococcal conjugates combined.

The Vaccine Composition

The composition used according to the invention will typically include apharmaceutically acceptable carrier. Such carriers include any carrierthat does not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolised macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, sucrose, trehalose, lactose, and lipidaggregates (such as oil droplets or liposomes). Such carriers are wellknown to those of ordinary skill in the art. The vaccines may alsocontain diluents, such as water, saline, glycerol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable carriersand excipients is available in reference 78.

Compositions used according to the invention may include anantimicrobial, particularly if packaged in a multiple dose format.

Compositions used according to the invention may comprise detergent e.g.a TWEEN™ (polysorbate), such as TWEEN 80™. Detergents are generallypresent at low levels <0.01%.

Compositions used according to the invention may include sodium salts(e.g. sodium chloride and/or sodium phosphate). These can be used fortonicity. A concentration of 10±2 mg/ml NaCl is typical e.g. about 8.8mg/ml. A concentration of 1.2 mg/ml sodium phosphate is typical.

Compositions used according to the invention will generally include abuffer e.g. a phosphate buffer.

Compositions used according to the invention may comprise a sugaralcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose)e.g. at about 15-30 mg/ml (e.g. 25 mg/ml), particularly if they are tobe lyophilised or if they include material which has been reconstitutedfrom lyophilised material. Preferred compositions, however, are notlyophilised i.e. all meningococcal conjugates are present in aqueousform, from the packaging stage to the administration stage.

Compositions will generally be administered directly to a patient.Direct delivery may be accomplished by parenteral injection (e.g.subcutaneously, intraperitoneally, intravenously, intramuscularly, or tothe interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration (e.g. to the thighor the upper arm) is preferred. Injection may be via a needle (e.g. ahypodermic needle), but needle-free injection may alternatively be used.A typical intramuscular dose is 0.5 ml.

Meningococcal conjugates from multiple serogroups are administered inadmixture within a single composition. The composition may beadministered as a single dose, or may be administered more than once ina multiple dose schedule. Multiple doses may be used in a primaryimmunisation schedule and/or in a booster immunisation schedule. Aprimary dose schedule may be followed by a booster dose schedule of themeningococcal conjugates. Suitable timing between priming doses (e.g.between 4-16 weeks), and between priming and boosting, can be routinelydetermined. The conjugates may conveniently be administered at the sametime as other vaccines e.g. at the same time as a D-T-P vaccine, or atthe same time as a pneumococcal conjugate vaccine, or at the same timeas an influenza vaccine, or at the same time as a MMR or MMRV vaccine.These vaccines will generally be administered separately but during thesame visit to the doctor.

Bacterial infections can affect various areas of the body and socompositions may be prepared in various forms. For example, thecompositions may be prepared as injectables, either as liquid solutionsor suspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared (e.g. alyophilised composition). The composition may be prepared for topicaladministration e.g. as an ointment, cream or powder. The composition beprepared for oral administration e.g. as a tablet or capsule, or as asyrup (optionally flavoured). The composition may be prepared forpulmonary administration e.g. as an inhaler, using a fine powder or aspray. The composition may be prepared as a suppository or pessary. Thecomposition may be prepared for nasal, aural or ocular administratione.g. as spray, drops, gel or powder [e.g. refs 79 & 80]. In general,however, the meningococcal conjugates are formulated for intramuscularinjection.

Compositions used according to the invention may or may not include avaccine adjuvant. Adjuvants which may be used in compositions of theinvention include, but are not limited to:

A. Mineral-containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. see chapters 8 & 9 of ref. 81], or mixtures ofdifferent mineral compounds, with the compounds taking any suitable form(e.g. gel, crystalline, amorphous, etc.), and with adsorption beingpreferred. The mineral containing compositions may also be formulated asa particle of metal salt [82].

Aluminium phosphates are particularly preferred, and a typical adjuvantis amorphous aluminium hydroxyphosphate with PO₄/Al molar ratio between0.84 and 0.92, included at about 0.6 mg Al³⁺/ml. Adsorption with a lowdose of aluminium phosphate may be used e.g. between 50 and 100 μg Al³⁺per conjugate per dose. Where a composition includes conjugates frommultiple bacterial species then not all conjugates need to be adsorbed.

B. Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 [Chapter 10 of ref. 81;see also ref. 83] (5% Squalene, 0.5% TWEEN 80™, and 0.5% SPAN 85™,formulated into submicron particles using a microfluidizer). CompleteFreund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may alsobe used.

C. Saponin Formulations [Chapter 22 of Ref 81]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs. QS21 is marketed as Stimulon™.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref. 84.Saponin formulations may also comprise a sterol, such as cholesterol[85].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs) [chapter 23 ofref. 81]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA & QHC. ISCOMs are further described in refs. 85-87. Optionally, theISCOMS may be devoid of additional detergent [88].

A review of the development of saponin based adjuvants can be found inrefs. 89 & 90.

D. Virosomes and Virus-like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or, isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 91-96. Virosomes are discussed further in, for example, ref.97.

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacteriallipopolysaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 98. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane [98]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [99,100].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 101 & 102.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 103, 104 and 105 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 106-111.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [112]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 113-115. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, refs. 112 & 116-118.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 119 and as parenteraladjuvants in ref 120. The toxin or toxoid is preferably in the form of aholotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 121-128. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref. 129, specificallyincorporated herein by reference in its entirety.

F. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [130], etc.) [131], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor.

G. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [132] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [133].

H. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

I. Liposomes (Chapters 13 & 14 of Ref 81)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 134-136.

J. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [137]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [138] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [139]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

L. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include —N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

K Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 140 and 141.

M. Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 142 and 143.

N. Thiosemicarbazone Compounds.

Examples of thiosemicarbazone compounds, as well as methods offormulating, manufacturing, and screening for compounds all suitable foruse as adjuvants in the invention include those described in ref. 144.The thiosemicarbazones are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

O. Tryptanthrin Compounds

Examples of tryptanthrin compounds, as well as methods of formulating,manufacturing, and screening for compounds all suitable for use asadjuvants in the invention include those described in ref. 145. Thetryptanthrin compounds are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention: (1) a saponin and anoil-in-water emulsion [146]; (2) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL) [147]; (3) a saponin (e.g. QS21)+a non-toxic LPSderivative (e.g. 3dMPL) +a cholesterol; (4) a saponin (e.g.QS21)+3dMPL+IL-12 (optionally+a sterol) [148]; (5) combinations of 3dMPLwith, for example, QS21 and/or oil-in-water emulsions [149]; (6) SAF,containing 10% squalane, 0.4% TWEEN 80™, 5% PLURONIC-BLOCK POLYMERL121™, and thr-MDP, either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion. (7) RIBI™ adjuvantsystem (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% TWEEN 80™,and one or more bacterial cell wall components from the group consistingof monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS ( DETOX™); (8) one or more mineralsalts (such as an aluminum salt)+a non-toxic derivative of LPS (such as3dMPL); and (9) one or more mineral salts (such as an aluminum salt)+animmunostimulatory oligonucleotide (such as a nucleotide sequenceincluding a CpG motif).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 81.

The use of an aluminium hydroxide or aluminium phosphate adjuvant isparticularly preferred, and conjugates are generally adsorbed to thesesalts [e.g. examples 7 & 8 of ref. 9; example J of ref. 10]. Mixing withaluminium salts with no adsorption is also possible [27, 76]. Calciumphosphate is another preferred adjuvant. Conjugates may be mixed with(and optionally adsorbed to) the adjuvants separately and then theconjugates may be mixed together, or the conjugates may be mixedtogether and then mixed with adjuvant.

The pH of compositions used according to the invention is preferablybetween 6 and 8, preferably about 7. Stable pH may be maintained by theuse of a buffer. Where a composition comprises an aluminium hydroxidesalt, it is preferred to use a histidine buffer [150]. The compositionmay be sterile and/or pyrogen-free. Compositions may be isotonic withrespect to humans.

Compositions may include a preservative (e.g. thiomersal,2-phenoxyethanol), or may be preservative-free. Preferred compositionsof the invention do not include any mercurial material e.g. they arethiomersal-free.

To prevent interference between antigens, particularly conjugateantigens, it is possible to include a polyanionic polymer, such aspoly-L-glutamic acid [151].

Compositions may be presented in vials, or they may be presented inready-filled syringes. The syringes may be supplied with or withoutneedles. A syringe will include a single dose of the composition,whereas a vial may include a single dose or multiple doses. Injectablecompositions will usually be liquid solutions or suspensions.Alternatively, they may be presented in solid form (e.g. freeze-dried)for solution or suspension in liquid vehicles prior to injection.

Compositions may be packaged in unit dose form or in multiple dose form.For multiple dose forms, vials are preferred to pre-filled syringes.Effective dosage volumes can be routinely established, but a typicalhuman dose of the composition for injection has a volume of 0.5 ml.

Where a composition is to be prepared extemporaneously prior to use(e.g. where a component is presented in lyophilised form) and ispresented as a kit, the kit may comprise two vials, or it may compriseone ready-filled syringe and one vial, with the contents of the syringebeing used to reactivate the contents of the vial prior to injection.For compositions that include a serogroup A capsular saccharide then theserogroup A saccharide may be lyophilised, whereas saccharide(s) fromother serogroup(s) may be present in liquid form.

Compositions will comprise an immunologically effective amount of themeningococcal conjugates, as well as any other components, as needed. By‘immunologically effective amount’, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, elicits a protective anti-meningococcal immune response inpatients. This amount varies depending upon; the health and physicalcondition of the individual to be treated, age, the taxonomic group ofindividual to be treated (e.g. non-human primate, primate, etc.), thecapacity of the individual's immune system to synthesise antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine, trials,and a typical quantity of each meningococcal antigen per dose is between1 μg and 20 μg per serogroup (measured in terms of saccharide) e.g.between 2 and 10 μg per serogroup. A dose of about 4 μg per serogroup ispreferred (i.e. a total of 16 μg in a tetravalent mixture).

The total amount of carrier protein in a composition preferably does notexceed 100 μg/dose e.g. it is ≦90 μg/dose, ≦80 μg/dose, ≦70 μg/dose, ≦60μg/dose, ≦50 μg/dose, etc. The total amount of carrier protein in acomposition will generally be at least 10 μg/dose.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

MODES FOR CARRYING OUT THE INVENTION

Lack of Carrier Suppression Using Tetravalent A/C/W135/Y ConjugateMixture

Mixtures of meningococcal conjugates for serogroups A+C, C+W+Y orA+C+W+Y can be prepared as described in references 9 and 10. If desired,these can be mixed with aluminium hydroxide or aluminium phosphateadjuvants, as also described in references 9 and 10. These vaccines haveeither CRM197 or diphtheria toxoid (Dt) as the carrier protein,covalently linked to the saccharides. Patients who received pediatricD-T-P vaccination (either D-T-Pa or D-T-Pw), including those whoreceived vaccines containing D-T-P and other antigens (e.g. D-T-P-Hibtetravalent, D-T-P-HBsAg tetravalent, D-T-P-Hib-HBsAg pentavalent,D-T-P-Hib-HBsAg-IPV hexavalent, etc.) are selected to receive themixture of conjugates that has a Dt carrier. This tetravalent mixture ofconjugates is immunogenic in humans [9, 11, 13].

Patients who received pediatric Hib vaccination (either as a monovalentHib, with or without D-T-Pa or D-T-Pw; or as part of a combinationvaccine such as D-T-P-Hib tetravalent, D-T-P-Hib-HBsAg pentavalent,D-T-P-Hib-HBsAg-IPV hexavalent, etc.) are selected to receive themixture of conjugates that has a CRM197 carrier.

A control group of patients is selected to receive one of the twoconjugate mixtures. The control patients have not previously receivedeither diphtheria toxoid or CRM197, either as separate antigens or ascarrier proteins in conjugates.

The ability of the tetravalent conjugates to elicit an immune responsein the patients is assessed. Carrier suppression is indicated if thetest groups show significantly lower anti-meningococcal immune responsesthan the control patients, and in particular if the conjugates fail toelicit a useful SBA response in the patients.

In clinical trial V59P2, conducted in Finland and Germany with 620subjects aged 12-16 months, five formulations were tested. The vaccinesused CRM197 carrier and an aluminium phosphate adjuvant [10]. Doses ofeach serogroup saccharide, expressed as μg saccharide mass per 0.5 mldose, were as follows:

Group MenA MenC MenW135 MenY 1 10 10 10 10 2 0 10 10 10 3 10 5 5 5 4 5 55 5 5 2.5 2.5 2.5 2.5

Subjects received an injection at time zero, and 25% of the subjectsthen received a second dose of the vaccine 4 weeks later.

Sera of patients were collected 1 month after vaccine administration andwere tested in a SBA assay against N. meningitidis from each serogroup,using human complement. SBA titre increase relative to time zero serawas assessed, with criteria being ≧1:4 and ≧1:8. Anti-capsule titres(GMT) were also measured for each serogroup. Results are shown in Table1 below.

Thus the trivalent and tetravalent vaccines were both immunogenic intoddlers. The conjugates are immunogenic at saccharide doses as low as2.5 μg per conjugate. The immune response are boostable, with large SBAtitre increases after the second dose. No evidence of carriersuppression was seen in this trial.

Lack of Suppression of Anti-Dt Responses

A Belgian study from 1999 [26] demonstrated impaired tetanus immunity ininfants, persisting into early childhood, following receipt of a primarycourse of Hib vaccine conjugated to tetanus toxoid. The conjugate'scarrier protein was therefore having a negative immunological impact. Incontrast, the opposite effect was seen in a study of pneumococcalconjugate vaccines [152]. The possibility that a meningococcal conjugatemight interfere with diphtheria immunity [153] was studied using aconjugate of meningococcal serogroup C saccharide to a CRM197 carrier(Menjugate™).

Children were split into five groups, who were immunised in the firstyear of life as follows: (1) four doses of Menjugate™; (2) three dosesof Menjugate™, plus one dose of a bivalent unconjugated A/C mixture; (3)three doses of HBsAg vaccine then one dose of Menjugate™; (4) threedoses of HBsAg vaccine then one dose of a bivalent unconjugated A/Cmixture; (5) controls to receive no meningococcal vaccines. All childrenalso received three doses of diphtheria vaccine during the first fourmonths of life, but had not received a pre-school diphtheria booster.

Patients received a single dose of Menjugate™ at 4 years of age, andblood was sampled pre- and post-(˜30 days) this Menjugate™injection.Diphtheria antibodies were measured by ELISA and geometric meanconcentrations were assessed. The percentage of subjects with anantibody level ≧0.1 IU/ml was also assessed. Results were as follows:

Pre-vaccination Post-vaccination Group GMC % ≧ 0.1 GMC % ≧ 0.1 1 0.3594% 9.00 100% 2 0.18 87% 8.55 100% 3 0.20 81% 2.82 100% 4 0.10 51% 4.5599%

The baseline anti-Dt titres were higher in patients who had previouslyreceived Menjugate™ than in those who had not (e.g. compare groups 1 and4). Simple regression analysis revealed significant linear relationshipsbetween the number of previous Menjugate™ doses (4, 3, 1 and 0 forgroups 1, 2, 3 and 4, respectively) and (a) pre-vaccination anti-Dttitres and (b) post-vaccination anti-Dt titres.

Thus there is enhanced persistence of immunity to diphtheria at 4 yearsof age in children who received four Menjugate™ doses in infancy.Furthermore, there is a trend towards higher anti-Dt antibody responsesfollowing a booster dose of Menjugate™ in patients who had received atleast three Menjugate™ doses as infants. No evidence of immunologicalinterference between meningococcal conjugates and diphtheria immunitywas found.

Bivalent A/C Conjugate Mixture Shows No Interference with DTP

A mixture of capsular saccharides from serogroups A and C [8] has beenadministered to infants (5 to 11 weeks old) in Niger [154] who had notpreviously received DTP vaccine. Children received either unconjugatedsaccharides (50 μg of each serogroup) or unadjuvanted Dt-conjugatedsaccharides (4 μg of each), with six different schedules:

-   -   (1) Four conjugate doses: 6 weeks, 10 weeks, 14 weeks, 9 months.    -   (2) Three conjugate doses: 6 weeks, 10 weeks, 14 weeks.    -   (3) Two conjugate doses: 14 weeks, 9 months.    -   (4) One conjugate doses: 14 weeks.    -   (5) One conjugate dose: 9 months.    -   (6) One unconjugated dose: 9 months.

The children received DTP and oral polio vaccines at 6, 10 and 14 weeks,with a booster at 9 months, and the meningococcal vaccines wereadministered at the same time as these existing vaccines. To assessanamnestic responses, the children were also given an unconjugatedvaccine at 24 months.

Serum bactericidal antibody responses were measured at 18 weeks (i.e. 4weeks after the third DTP/polio vaccination), at 10 months: (i.e. 1month after the 9 month DTP/polio booster) and 1 week after theadministration of unconjugated material at 24 months. The percentages ofpatients showing a ≧128-fold increase in SBA titres were as follows:

% 18 weeks 10 months 24¼ months Schedule MenA MenC MenA MenC MenA MenC(1) 56 84 89 73 100 95 (2) 56 86 6 9 96 82 (3) 68 64 85 85 100 95 (4) 6157 4 8 100 93 (5) 3 7 62 26 100 99 (6) 2 2 5 11 97 52

There was no difference in antibody responses against diphtheria toxoidbetween the six groups [8].

Thus there is no evidence of interference resulting from the use ofdiphtheria toxoid as both a protective antigen and as a carrier for theconjugates. For example, patients in group 5 had received diphtheriatoxoid in DTP vaccines at 6, 10 and 14 weeks before receiving the firstdose of meningococcal conjugate, but showed an anti-meningococcal SBAresponse of >99% at 24 months.

No Negative Impact on Anti-diphtheria Responses

As mentioned above, patients receiving meningococcal conjugate vaccinesat the same time as DTP vaccines showed no reduction in immune responsesagainst diphtheria toxoid. In another study, meningococcal conjugatesand DTP have been administered at different times. A three dose DTPschedule at 2, 3 and 4 months of age had been followed by a single doseof a bivalent A/C vaccine either with Dt-conjugated saccharides or withunconjugated saccharides. Immune responses against diphtheria toxoidwere measured by ELISA, and GM titres were as follows:

Antigens Pre-immunisation Post-immunisation Conjugated 0.05 21.2Unconjugated 0.06 0.06

The unconjugated saccharides did not cause any anti-Dt response(unsurprisingly), but the conjugated saccharides resulted in a stronganti-DT response. Administration of these conjugates may thus provideanti-diphtheria immunity in naïve patients, or may take the place of aDt booster.

It will be understood that the invention is described above by way ofexample only and modifications may be made while remaining within thescope and spirit of the invention.

TABLE 1 Results of trial V59P2 Group A C W135 Y GMT (1 month after 1dose) 1 3.9 6.4 7.1 8.9 2 2 6.1 8.3 8.5 3 5.7 5.2 6.9 12 4 3.8 4.5 7.09.6 5 3.9 5.3 7.0 12 GMT (1 month after 2 doses) 1 27 89 22 37 2 2 80 2057 3 29 76 28 58 4 14 47 20 35 5 17 71 23 52 % patients with SBA ≧ 1:4(1 month after 1 dose) 1 33 56 57 58 2 0 57 60 61 3 55 49 53 70 4 37 4254 64 5 40 51 57 67 % patients with SBA ≧ 1:4 (1 month after 2 doses) 1100 100 96 96 2 0 100 73 92 3 91 96 95 95 4 84 96 88 96 5 80 100 80 92 %patients with SBA ≧ 1:8 (1 month after 1 dose) 1 25 44 46 48 2 0 40 5049 3 39 34 45 64 4 23 30 44 51 5 26 35 40 60 % patients with SBA ≧ 1:8(1 month after 2 doses) 1 92 100 85 93 2 0 100 64 92 3 87 96 95 82 4 6092 77 92 5 72 92 72 88References (The Contents of which are Hereby Incorporated by Reference)

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The invention claimed is:
 1. A method of immunising a human patientagainst Neisseria meningitides, comprising the step of administering tothe human patient a composition that comprises at least two of (a) aconjugate of (i) the capsular saccharide of serogroup A N. meningitidisand (ii) a diphtheria toxoid carrier or CRM197 carrier; (b) a conjugateof (i) the capsular saccharide of serogroup C N. meningitidis and (ii) adiphtheria toxoid or CRM197 carrier; (c) a conjugate of (i) the capsularsaccharide of serogroup W135 N. meningitidis and (ii) a diphtheriatoxoid or CRM197 carrier; and (d) a conjugate of (i) the capsularsaccharide of serogroup Y N. meningitidis and (ii) a diphtheria toxoidor CRM197 carrier, wherein the patient has been pre-immunised with avaccine comprising (x) a non-conjugated diphtheria toxoid and (z) aconjugate of (i) a capsular saccharide of an organism other than N.meningitidis and (ii) a diphtheria toxoid or CRM197 carrier, wherein thepatient was pre-immunised at least six months before the immunizationwith the N. meningitidis conjugates.
 2. The method of claim 1, whereinthe composition comprises all four of (a), (b), (c) and (d).
 3. Themethod of claim 1, wherein the organism other than the N. meningitidisis Haemophilus influenzae type B.
 4. The method of claim 1, wherein thepatient has been pre-immunised with a pneumococcal capsular saccharideconjugated to a diphtheria toxoid or CRM197 carrier.
 5. The method claim1, wherein the patient was pre-immunised at least eight years before themethod of immunization.
 6. The method of claim 1, wherein thepre-immunisation took place within one year of the patient's birth. 7.The method of claim 1, wherein the saccharides in the conjugates (a) to(d) are shorter than the native capsular saccharides of thecorresponding serogroups of N. meningitides.
 8. The method of claim 1,wherein the conjugates (a) to (d) comprise the diphtheria toxoid carrierand an adipic acid linker.
 9. The method of claim 8, wherein thecomposition comprises no more than 60 microgram of the diphtheria toxoidcarriers.
 10. The method of claim 1, wherein the conjugates (a) to (d)comprise the CRM197 carrier and an adipic acid linker.
 11. The method ofclaim 1, wherein the composition further comprises a conjugated capsularsaccharide from Streptococcus pneumoniae.
 12. The method of claim 1,wherein the composition further comprises a conjugated capsularsaccharide from Haemophilus influenzae type B.
 13. The method of claim1, wherein the composition further comprises a protein antigen fromserogroup B of Neisseria meningitides.
 14. The method of claim 1,wherein the composition includes an aluminium hydroxide adjuvant and/oran aluminium phosphate adjuvant.
 15. The method of claim 2, wherein thepatient has been pre-immunised with a vaccine comprising (x) and (z).16. The method of claim 2, wherein the organism other than the N.meningitidis is Haemophilus influenzae type B.
 17. The method of claim2, wherein the patient has been pre-immunised with a pneumococcalcapsular saccharide conjugated to a diphtheria toxoid or CRM197 carrier.18. The method claim 2, wherein the patient was pre-immunised at leasteight years before the method of immunization.
 19. The method of claim2, wherein the pre-immunisation took place within one year of thepatient's birth.
 20. The method of claim 2, wherein the saccharides inthe conjugates (a) to (d) are shorter than the native capsularsaccharides of the corresponding serogroups of N. meningitides.
 21. Themethod of claim 2, wherein the conjugates (a) to (d) comprise thediphtheria toxoid carrier and an adipic acid linker.
 22. The method ofclaim 19, wherein the composition comprises no more than 60 microgram ofthe diphtheria toxoid carriers.
 23. The method of claim 2, wherein theconjugates (a) to (d) comprise the CRM197 carrier and an adipic acidlinker.
 24. The method of claim 2, wherein the composition furthercomprises a conjugated capsular saccharide from Streptococcuspneumoniae.
 25. The method of claim 2, wherein the composition furthercomprises a conjugated capsular saccharide from Haemophilus influenzaetype B.
 26. The method of claim 2, wherein the composition furthercomprises a protein antigen from serogroup B of Neisseria meningitides.27. The method of claim 2, wherein the composition includes an aluminiumhydroxide adjuvant and/or an aluminium phosphate adjuvant.